To Construct an Engineered ( S )-Equol Resistant E. coli for in Vitro ( S )-Equol Production.

( S )-equol is one of the major metabolites of daidzein that is produced by human and animal gut bacteria. Most of the physiological functions of soybean isoflavones, such as anti-oxidative activity, anti-cancer activity, and cardiovascular protection have been ascribed to ( S )-equol. However, only 30-50% people contain this kind of equol-producing bacteria, and therefore are able to convert daidzein to ( S )-equol. Administration of ( S )-equol may be more beneficial than soybean isoflavones. The aim of this study was to construct an engineered ( S )-equol resistant Escherichia coli to enhance ( S )-equol production in vitro . First, transposon mutagenesis libraries were constructed and screened to isolate the ( S )-equol resistant mutant E. coli strain BL21 ( ydiS ) in order to overcome the inhibitory effects of ( S )-equol on bacterial growth. Bacterial full genome scan sequencing and in vitro overexpression results revealed that the ydiS gene was responsible for this resistance. Second, the ( S )-equol-producing genes L- dznr , L- ddrc , L- dhdr , and L- thdr of Lactococcus strain 20-92 were synthesized and cloned into compatible vectors, pETDuet-1 and pCDFDuet-1. These plasmids were subsequently transformed into BL21 (DE3) and its mutant BL21 ( ydiS ). Both engineered BL21 (DE3) and BL21 ( ydiS ) could use daidzein as substrate to produce ( S )-equol under both anaerobic and aerobic conditions. As expected, engineered BL21 ( ydiS ) had faster growth rates than BL21 (DE3) when supplemented with high concentrations of ( S )-equol. The yield and the daidzein utilization ratio were higher for engineered BL21 ( ydiS ). Interestingly, engineered BL21 ( ydiS ) was able to convert daidzein to ( S )-equol efficiently under aerobic conditions, providing a convenient method for ( S )-equol production in vitro . In addition, a two-step method was developed to produce ( S )-equol using daidzin as substrate.